Hi-C derived insulation strength predicts evolutionary conservation of topologically associating domains

Topologically associating domains (TADs) are fundamental units of three-dimensional chromatin organization, yet their functional significance and evolutionary conservation remain debated. We generate bulk Hi-C contact maps from the ventral hippocampus of eight inbred mouse strains, including five classical laboratory strains and three wild-derived strains, comprising 14.55 billion valid contacts. We show that discrete TAD and loop calling yields poor inter-replicate concordance, whereas separation scores, a continuous measure of boundary insulation strength, substantially improve reproducibility. Using this approach, we identify 2,857 highly insulated genomic regions whose insulation is conserved across all strains. These regions are enriched for multiple CTCF binding sites, accessible chromatin, and proximity to super-enhancers and housekeeping genes, and show greater DNA sequence conservation across placental mammals and more stable DNA methylation than expected by chance. Cross-species analysis reveals that the positional overlap of insulated regions between human and mouse increases with insulation strength, reaching 80.7% for the most highly insulated regions. While genomic structural rearrangements are the primary driver of chromatin conformation differences between strains, we find limited evidence that these differences have functional consequences. Together, these findings show that insulation strength tracks the evolutionary conservation of chromatin domain boundaries and correlates with features associated with functional importance, providing a quantitative framework for prioritizing candidate functionally critical TAD boundaries.